PROJECT SUMMARY Variation in the craniofacial skeleton produces both a spectrum of unique faces and as well as clinical malformations. Congenital craniofacial anomalies are one of the most common birth defects. In addition to genetic changes, we are increasingly recognizing the importance of the physical packaging of DNA in the regulation of gene expression and development, and the resulting genotype-phenotype relationship. A major determinant of this epigenetic regulation is post-translational modifications of histone proteins, which alters the structure of chromatin and access to DNA. The goal of this work is to determine how variation in chromatin structure, in particular histone acetylation, impacts phenotypic variation of the facial skeleton. We will capitalize on the unparalleled natural craniofacial variation among the evolutionary radiation of non-model cichlid fishes. Given that cichlid facial variation mimics human facial variation and the molecular control of facial development is conserved across vertebrates, this may yield novel insights into the epigenetic basis of variation in human faces. In Aim 1, we will comprehensively quantify variation in chromatin structure and DNA accessibility using ATAC-seq (Assay for Transposase Accessible Chromatin) and the downstream transcriptional effects using RNA-seq. These genomic analyses will be conducted in three cichlid species with diverse adult morphologies at three developmental time points key for facial divergence. In Aim 2, we will use pharmacological agents to alter the activity of proteins associated with histone acetylation during distinct windows of facial development. We will focus on four drugs that are potential sources of birth defects in humans; these drugs are in clinical trials for treatment of diseases from cancer to heart disease, but the FDA does not currently note a potential risk for pregnant women. Completion of this aim will determine windows of sensitivity for epigenetic changes and potential teratogenic effects of these drugs. Applying these drugs in closely-related cichlids (comparable to comparing different mouse strains), allows investigation of gene by environment (GxE) interactions previously demonstrated for similar drugs. This work will address a major gap in our knowledge of the genotype- phenotype relationship, the role of epigenetic regulation in phenotypic variation. Application of drugs to cichlid fishes enables discovery of genetic interactions that may define sensitivity to exposure and risk of craniofacial malformations in humans. In line with the goals of the R15 AREA grant, undergraduates will conduct bioinformatic analyses in Aim 1, and lead pharmacological manipulations in Aim 2. Grant funds will support four new undergraduate researchers, including summer fellowships that enable a full immersion in research without the constraints of a full class schedule.